Methods for coupling targeting peptides onto recombinant lysosomal enzymes for improved treatments of lysosomal storage diseases

ABSTRACT

Described herein are methods of making targeting peptides conjugated to a recombinant lysosomal enzyme by modifying the amino (N)-terminus and one or more lysine residues on a recombinant human lysosomal enzyme using a first crosslinking agent to give rise to a first crosslinking agent modified recombinant human lysosomal enzyme, modifying a lysine or cysteine within a short extension linker at the carboxyl (C)-terminus on a variant IGF-2 peptide having a short extension linker using a second crosslinking agent to give rise to a second crosslinking agent modified variant IGF-2 peptide, and then conjugating the first crosslinking agent modified recombinant human lysosomal enzyme to the second crosslinking agent modified variant IGF-2 peptide containing a short extension linker. Also described herein are conjugates synthesized using the methods disclosed herein. Also described herein are treatment methods using the disclosed conjugates.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 14/122,858filed Jun. 2, 2014, which is the National Stage of InternationalApplication No. PCT/US2012/039705 filed May 25, 2012, which claims thebenefit of U.S. Provisional Application No. 61/490,957, filed May 27,2011, both of which are incorporated by reference herein in itsentirety.

TECHNICAL FIELD

The technical field relates to peptide chemistry. The technical fieldalso relates to targeting of recombinant lysosomal enzymes to thelysosome in the treatment of lysosomal storage diseases.

BACKGROUND

Lysosomes are specialized intracellular organelles where proteins,various lipids (including glycolipids and cholesterol) and carbohydratesare degraded and recycled to their primary constituents that enablesynthesis of new proteins, membrane components and other molecules.Lysosomes are also utilized by cells to help maintain homeostasis andcellular health through an adaptive cellular process known as autophagythat increases lysosomal activity to provide additional amino acids forincreased biosynthesis of various proteins (e.g., antibodies andinterferons) and to supply nutrients for energy production to deal withstressful periods of nutrient deprivation or viral infections. Eachmetabolic process is catalyzed by a specific resident lysosomal enzyme.Genetic mutations can cause deficiencies in lysosomal biologicalactivities that alter metabolic processes and lead to clinical diseases.Lysosomal storage disorders (LSDs) are a class of approximately 50different human metabolic diseases caused by a deficiency for specificlysosomal proteins that results in the accumulation of varioussubstances within the endosomal/lysosomal compartments. Many of thesediseases have been well-characterized to understand the deficientlysosomal protein and the resultant metabolic defect. For example, thereare several LSDs of altered glycolipid catabolism such as Gaucher,Fabry, and Tay-Sachs/Sandhoff. Neimann-Pick C is characterized byimpaired lipid and cholesterol metabolism while diseases of alteredcarbohydrate metabolism such as glycogen storage diseases type II(Pompe) and type III (Corey-Forbes) have also been characterized. OtherLSDs alter metabolism of bone or extracellular matrices [e.g.,mucopolysaccharidoses (MPS I-VII), Gaucher] and protein turnover(neuronal ceroid lipofuscinoses; Batten, etc.). While LSDs arerelatively rare, they can cause severe chronic illness and often deathif not effectively treated.

There are no known cures for lysosomal storage diseases but a number ofdifferent treatment approaches have been investigated for various LSDsincluding bone marrow and umbilical cord blood transplantation, enzymereplacement therapy (ERT), substrate reduction therapy (SRT) andpharmacological chaperone therapy. Gene therapy is also being developedbut has not been tested clinically. Of these treatment approaches, ERTis the most established with multiple ERTs approved for the treatment ofvarious LSDs including Gaucher, Fabry, Pompe, MPS I, MPS II and MPS VIwhile one SRT drug is approved for the treatment of Gaucher disease.

The concept of ERT for the treatment of a lysosomal storage disease isfairly straightforward where a recombinant human lysosomal enzyme isadministered in patients to supplement the deficient biological activityand improve clinical symptoms. However, unlike other protein therapeutictreatments that function primarily at the cell surface or outside ofcells (e.g., anti-VEGF and other antibodies, erythropoietin, clottingfactors, etc.), lysosomal enzymes must function inside cells, withinlysosomes, and therefore require a mechanism for entering cells from theoutside and subsequent delivery to these internal compartments. Inmammals, the branched carbohydrate structures on the protein backbone oncertain asparagine residues (N-linked oligosaccharides; N-glycans) formost soluble lysosomal enzymes are post-translationally modified to forma specialized carbohydrate structure called mannose 6-phosphate (M6P).M6P is the natural biological signal for identification and transport ofnewly synthesized lysosomal proteins from the Golgi apparatus tolysosomes via membrane-bound M6P receptors. A class of M6P receptors(cation-independent M6P receptor; CI-MPR) also cycles to the plasmamembrane and is functionally active for binding and internalizingexogenous lysosomal proteins. The CI-MPR is believed to have evolved torecapture lysosomal proteins that escaped cells (via secretion out ofcells) and thus, provide a targeting mechanism for internalizingexogenous lysosomal proteins and is the basis for enzyme replacementtherapy for various LSDs.

Recombinant lysosomal enzyme replacement therapies have been shown to begenerally safe but their effectiveness for reducing clinical symptomsvaries widely. For example: Fabrazyme™ (recombinant acid α-galactosidaseA; Genzyme Corp.) ERT dosed at 1 mg/kg body weight every other week issufficient to clear accumulated substrate from endothelial cells inFabry disease while 40 mg/kg of Myozyme™ (recombinant human acidα-glucosidase, rhGAA; Genzyme Corp.) dosed every other week is onlymoderately effective for Pompe disease. The disparate efficacy isprimarily attributed to differences in the M6P content such that lowlevels of M6P correlates with poor drug targeting and lower efficacy.The manufacture of recombinant lysosomal enzymes is very challengingbecause it is extremely difficult to control carbohydrate processing,particularly the level of M6P in mammalian expression systems. Twospecialized Golgi enzymes catalyze the M6P modification;N-acetylglucosamine phosphotransferase adds phosphate-linkedN-acetylglucosamine onto certain terminal mannose residues whileN-Acetylglucosamine-1-phosphodiester α-N-acetylglucosaminidase (alsoknown as Uncovering Enzyme) removes the covering N-acetylglucosamine toreveal the M6P signal. However, N-acetylglucosamine phosphotransferaseis limiting in cells and this biochemical reaction is inherentlyinefficient for various lysosomal proteins. Over-expression of lysosomalproteins during the manufacturing process greatly exacerbates thisproblem and leads to highly variable amounts of M6P. Consequently,carbohydrate processing is typically incomplete and leads to theproduction of recombinant lysosomal enzymes with mixtures of N-glycansthat contain M6P, non-M6P structures of high-mannose type N-glycans andcomplex-type N-glycans (typical for secretory proteins). To complicatematters, dead or damaged cells release enzymes such as phosphatases intothe cell culture medium which remove M6P. Consequently, reduced M6Pcontent lowers the binding affinity of a recombinant lysosomal enzymefor M6P receptors and decreases its cellular uptake and thereby, reducedrug efficacy. Dead or damaged cells release other glycosidases thatremove other carbohydrates (e.g., sialic acids, galactose, etc.) toreveal internal carbohydrates that are not typically exposed and theseN-glycans are readily identified as aberrant. These incomplete N-glycanstructures increase the clearance rate of recombinant lysosomal proteinsfrom the circulation which can also reduce drug efficacy. Higher drugdoses are therefore necessary to compensate for reduced efficacy. Higherdrug dose requirements however have multiple negative implications: (1)higher drug dose could be cost-prohibitive by increasing an alreadyexpensive treatment; (2) high drug doses require long infusion times;(3) large amounts of circulating drug results in significant antibodyresponses (seen in most Pompe patients) and numerous patients have alsoexperienced allergic reactions during infusions. The FDA has issued a“black-label warning” for Myozyme and the drug is typically administeredvery slowly at the beginning but ramped up over the course of theinfusion. This strategy helps to mitigate the allergic responses butsignificantly lengthens infusion times where 12-hr infusions are notuncommon.

One potential strategy for improving drug targeting for variouslysosomal ERTs employs a targeting peptide to efficiently target ERTs tolysosomes without requiring the traditional M6P carbohydrate structures.This is conceptually feasible since the cation-independent M6P receptorcontains a distinct binding domain for a small peptide calledinsulin-like growth factor 2 (IGF-2) and this receptor is thereforeknown as the IGF-2/(IGF-2/CI-MPR). This receptor is in fact solelyresponsible for internalizing exogenous M6P-bearing lysosomal proteinsbecause the IGF-2/CI-MPR is present and biologically active on the cellsurface. The other class of M6P receptors, the cation-dependent M6Preceptor (CD-MPR), is only involved in the transport of lysosomalproteins within cells because it is not biologically active on cellsurfaces and lacks the IGF-2 peptide binding domain. The IGF-2/CI-MPRhas two separate binding sites for M6P (domains 1-3 and 7-9,respectively) such that it binds a mono-M6P N-glycan (1 M6P residue onN-glycan) with moderate affinity or a bis-M6P N-glycan (two M6P residueson the same N-glycan) with approximately 3000-fold higher affinity.Since lysosomal proteins contain mixtures of complex (no M6P), mono- andbis-M6P N-glycans, their affinities for the IGF-2/CI-MPR vary widelydepending on the type and amount of M6P-bearing N-glycans. The IGF-2peptide has the highest affinity for the IGF-2/CI-MPR that isapproximately 230,000-fold higher than the mono-M6P N-glycan. A summaryof the binding affinities of various ligands for the IGF-2/CI-MPR aresummarized below in Table 1.

TABLE 1 Ligand Affinity for IGF-2/CI-MPR Binding Affinity Ligand(Apparent Kd; nM) free M6P ^(a) 7000 pentamannose-M6P ^(a) 6000 bis-M6PN-Glycan ^(a) 2 beta-galactosidase ^(a) 20 WT hIGF-2 ^(b, c) 0.03-0.2[Leu27] hIGF-2 ^(c) 0.05 [Leu27] hIGF-2 ^(c) 0.06

In mammals, IGF-2 is the primary growth hormone during embryonicdevelopment. After birth, IGF-2 levels remain relatively constant eventhough it no longer mediates growth (growth mediated by IGF-1 viastimulation by human growth hormone throughout life). The role of IGF-2after birth is not well understood but this peptide is believed to aidwound healing and tissue repair. IGF-2 is mostly bound in thecirculation by serum IGF binding proteins (IGFBPs 1-6) which mediate thelevels of free IGF-2 peptide. These IGFBPs also bind insulin and IGF-1and regulate their circulating levels. The IGF-2/CI-MPR is the naturalclearance pathway for free IGF-2 peptide. Because IGF-2 is structurallysimilar to insulin and IGF-1, it has low affinity for the insulinreceptor (˜100-fold lower) and IGF-1 receptor (˜230-fold lower) comparedto the IGF-2/CI-MPR. This specificity can be improved considerably byeliminating various amino acids or substituting specific amino acidresidues (e.g., [Leu27] IGF-2 & [Leu43] IGF-2) to maintain high-affinitybinding to the IGF-2/CI-MPR (Table 1) but significantly decrease oreliminate binding to the insulin and IGF-1 receptors. Similarly, IGF2variants lacking the initial six amino acid residues or a substitutionof arginine for glutamic acid at position 6 has been shown tosignificantly reduce affinity of IGF2 peptide for IGFBPs. Importantly,IGF-2 peptide has been shown to be safe in clinical trials and isutilized clinically to help treat certain growth deficiencies. Thesecollective data suggest that the IGF-2 peptide potentially could beutilized as a targeting motif instead of the traditional M6Pcarbohydrate structures to facilitate the cellular uptake and transportof recombinant lysosomal enzymes to lysosomes.

There remains a need to develop strategies to create IGF-2-linkedproteins for improved protein targeting while overcoming carbohydrateprocessing issues.

SUMMARY

Provided herein are methods of making a targeting peptide conjugated toa recombinant lysosomal enzyme comprising modifying the amino(N)-terminus and one or more lysine residues on a recombinant humanlysosomal enzyme using a first crosslinking agent to give rise to afirst crosslinking agent modified recombinant human lysosomal enzyme,modifying the first amino acid of a short extension linker at the amino(N)-terminus on a variant IGF-2 peptide using a second crosslinkingagent to give rise to a second crosslinking agent modified variant IGF-2peptide, and then conjugating the first crosslinking agent modifiedrecombinant human lysosomal enzyme to the second crosslinking agentmodified variant IGF-2 peptide containing a short extension linker.

Also provided herein are methods of making a targeting peptideconjugated to a recombinant lysosomal enzyme comprising conjugating afirst crosslinking agent modified recombinant human lysosomal enzyme toone or more second crosslinking agent modified variant IGF-2 peptideswhere the first crosslinking agent modified recombinant lysosomal enzymecomprises a recombinant lysosomal enzyme characterized as having achemically modified N-terminus and one or more modified lysine residuesand the one or more second crosslinking agent modified variant IGF-2peptides comprise one or more variant IGF-2 peptides comprising amodified amino acid in a short linker at the amino (N)-terminus.

Provided herein are methods of making a molecule for enzyme replacementtherapy comprising conjugating a heterobifunctional crosslinking agentto a variant IGF-2 peptide and then conjugating the heterobifunctionalcrosslinking agent modified variant IGF-2 peptide to a recombinant humanlysosomal enzyme.

Also provided herein are methods of making a molecule for enzymereplacement therapy comprising conjugating a heterobifunctionalcrosslinking agent to a recombinant human lysosomal enzyme and thenconjugating the heterobifunctional crosslinking agent modifiedrecombinant human lysosomal enzyme to a variant IGF-2 peptide.

Provided herein are also conjugates comprising one or more variant IGF-2peptides chemically conjugated to a recombinant human lysosomal enzyme.

Conjugates comprising a heterobifunctional crosslinking agent modifiedvariant IGF-2 peptide conjugated to a recombinant human lysosomal enzymeare also provided.

Provided herein are methods for treating a subject suffering from alysosomal storage disease comprising administering to the subject aconjugate comprising one or more variant IGF-2 peptides chemicallyconjugated to a modified recombinant human lysosomal enzyme.

Also provided herein are methods for treating a subject suffering from alysosomal storage disease comprising administering to the subject aconjugate comprising a heterobifunctional crosslinking agent modifiedvariant IGF-2 peptide conjugated to a recombinant human lysosomalenzyme.

Also provided herein are methods of treating a patient suffering fromPompe, Fabry, Gaucher, MPS I, MPS II, MPS VII, Tay Sachs, Sandhoff,α-mannosidosis, or Wohlman disease comprising administering to a patientin need thereof, a composition comprising one or more variant IGF-2peptides chemically conjugated to a recombinant lysosomal enzyme and apharmaceutically acceptable carrier, in an amount sufficient to treatsaid disease.

Suitable methods of treating a patient suffering from Pompe, Fabry,Gaucher, MPS I, MPS II, MPS VII, Tay Sachs, Sandhoff, α-mannosidosis,Wohlman disease are also provided comprising administering to a patientin need thereof, a composition comprising a heterobifunctionalcrosslinking agent modified variant IGF-2 peptide conjugated to arecombinant human lysosomal enzyme and a pharmaceutically acceptablecarrier, in an amount sufficient to treat said disease.

Provided herein is DNA sequence that encodes a variant IGF-2 peptidethat was optimized for expression in E. coli comprising SEQ ID NO: 1.

Also provided herein are amino acids sequence that represents a variantIGF-2 peptide comprising SEQ ID NO: 2.

Amino acid sequences that represents an extension linker comprising SEQID NO: 3 are also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other aspects of the present invention is apparentfrom the following detailed description of the invention when consideredin conjunction with the accompanying drawings. For the purpose ofillustrating the invention, there is shown in the drawings embodimentsthat are presently preferred, it being understood, however, that theinvention is not limited to the specific instrumentalities disclosed.The drawings are not necessarily drawn to scale. In the drawings:

FIG. 1A shows a schematic for the conjugation of a hydrazide-modifiedlysosomal enzyme with a benzaldehyde-modified variant IGF2 peptide.Prior to this conjugation reaction, lysosomal enzymes are chemicallymodified with a first crosslinking agent such as N-succinimidyl6-hydrazinonicotinamide acetone (S-Hynic) which modifies the aminoterminus and one or more lysine residues on lysosomal enzymes tointroduce chemically active hydrazide functional groups. In a separatereaction, the N-terminal amino acid residue within a short extensionlinker region in a variant IGF2 peptide is chemically modified with asecond crosslinking agent such as PEG4-pentafluorobenzyne benzoate(PEG4-PFB) to introduce a benzaldehyde function group as described inpatent application. After purification of hydrazide-modified lysosomalenzymes and benzaldehyde-modified variant IGF2 peptides, these proteinsare incubated together in an acidic buffer containing aniline to formIGF2 peptide-conjugated lysosomal enzymes. In this conjugation reaction,chemically active hydrazide chemical groups react with aldehyde groupsto form stable covalent (hydrazone) linkages. FIG. 1B shows othersuitable first crosslinking agents (succinimidyl 6-hydrazinonicotinateacetone (S-Hynic), succinimidyl 4-hydrazidoterephthalate hydrochloride(SHTH), succinimidyl 4-hydrazinium nicotinate hydrochloride (SHNH), andN-hydroxysuccinimide ester-(PEG)n-hydrazide; wherein n=3-24 PEG units)and second crosslinking agents (PEG4-pentafluorobenzyne benzoate(PEG4-PFB), succinimidyl 4-formylbenzoate (SFB), and C6-succinimidyl4-formylbenzoate (C6-SFB)) that can be used.

FIG. 2A shows a schematic for the conjugation of phosphine-modifiedlysosomal enzyme with azide-modified variant IGF2 peptide via theStaudinger ligation reaction. Prior to this conjugation reaction,lysosomal enzymes are chemically modified with a first crosslinkingagent such as sulfo-NHS-phosphine which modifies the amino terminus andone or more lysine residues on lysosomal enzymes to introduce chemicallyactive phosphine functional groups. In a separate reaction, theN-terminal amino acid residue within a short extension linker region invariant IGF2 peptide is chemically modified with a second crosslinkingagent such as NHS-(PEG)n-azide to introduce an azide functional group.After purification of phosphine-modified lysosomal enzymes andazide-modified variant IGF2 peptide, these proteins are incubatedtogether in a slightly acidic buffer to form IGF2 peptide-conjugatedlysosomal enzymes. In this conjugation reaction, chemically active azidechemical groups react with phosphine groups to form stable covalent(amide) linkages. FIG. 2B shows other suitable first crosslinking agents(N-hydroxysuccinimide ester-phosphine (NHS-phosphine) andSulfo-N-hydroxysuccinimide ester-phosphine (Sulfo-NHS-phosphine) andsecond crosslinking agents (N-hydroxysuccinimide ester-azide(NHS-azide), N-hydroxysuccinimide ester-(PEG)n-azide; wherein n=3-24 PEGunits, and NHS-PEG3-S-S-azide) that can be used.

FIG. 3A shows a schematic for the conjugation of acetylene-modifiedlysosomal enzyme with azide-modified IGF2 peptide via Click chemistry.Prior to this conjugation reaction, lysosomal enzymes are chemicallymodified with a first crosslinking agent such as NHS-(PEG)n-acetylenewhich modifies the amino terminus and one or more lysine residues onlysosomal enzymes to introduce chemically active acetylene functionalgroups. In a separate reaction, the N-terminal amino acid residue withina short extension linker region in variant IGF2 peptide is chemicallymodified with a second crosslinking agent such as NHS-(PEG)n-azide tointroduce an azide functional group. After purification ofacetylene-modified lysosomal enzymes and azide-modified IGF2 peptide,these proteins are incubated together in slightly acidic buffer withcopper (I) ions to form IGF2 peptide-conjugated lysosomal enzymes. Inthis conjugation reaction, chemically active azide chemical groups reactwith alkyne groups to form stable covalent (triazole) linkages. FIG. 3Bshows other suitable first crosslinking agents (N-hydroxysuccinimideester-tetraoxapentadecane acetylene (NHS-PEG4-acetylene),N-hydroxysuccinimide ester-(PEG)n-acetylene; wherein n=3-24 PEG units,and NHS-PEG3-S-S-acetylene) and second crosslinking agents(N-hydroxysuccinimide ester-azide (NHS-azide), N-hydroxysuccinimideester-(PEG)n-azide; wherein n=3-24 PEG units, and NHS-PEG3-S-S-azide)that can be used.

FIG. 4A shows a schematic of conjugation of lysosomal enzymes and IGF2peptide using a single crosslinking agent such asm-maleimidobenzyol-N-hydroxysuccinimide ester (MBS). In the firstreaction, the chemically reactive maleimide group reacts with the freesulfhydryl group of a C-terminal cysteine reside in a IGF2 peptidevariant. The MBS-modified IGF2 peptide is then purified and thenconjugated to lysosomal enzymes via crosslinking of the chemicallyreactive N-hydroxysuccinimide ester group with the amino terminus andone or more lysine residues on lysosomal enzymes to form stable covalent(amide) linkages. FIG. 4B shows other suitable crosslinking agents(m-Maleimidobenzyol-N-hydroxysuccinimide ester (MBS),Sulfo-m-maleimidobenzyol-N-hydroxysuccinimide ester (sulfo-MBS), andSulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC))that can be used.

FIGS. 5A-5C show characterization of IGF2 peptides by C4 reverse phasechromatography. A 4.6×150 mm C4 reverse phase analytical column wasutilized for evaluating the purity and protein conformation of wildtypeand variant IGF2 peptides. Peptide samples were loaded onto C4 columnequilibrated with 0.1% trifluoracetic acid (TFA) and 25% acetonitrile.After 2 minutes, the column was developed using a 25-35% acetonitrilelinear gradient over a 10 min. FIG. 5A shows recombinant wildtype humanIGF2 peptide elutes at approximately 7.5 min corresponding to 30%acetonitrile. FIG. 5B shows recombinant variant human IGF2 peptide alsoelutes at approximately 7.5 min corresponding to 30% acetonitrile. FIG.5C shows PEG4-PFB modified variant human IGF2 peptide elutes atapproximately 8 min corresponding to 31% acetonitrile. These dataindicate that wildtype and variant IGF2 peptides have very similarprotein conformations since they behave nearly identical on C4 reversephase chromatography. The shift in retention time for PEG4-PFB modifiedvariant human IGF2 peptide indicates that the variant IGF2 peptide hadbeen completely modified with the chemical crosslinker which altered itsinteraction on the C4 column.

FIGS. 6A and 6B show evaluation of variant IGF2 peptide-conjugated rhGAAfor receptor binding and cellular uptake. Variant IGF2 peptide wasmodified with the crosslinker PEG4-PFB and subsequently coupled toS-Hynic-modified rhGAA. The resultant variant IGF2 peptide-conjugatedrhGAA (designated as vIGF2-rhGAA) was then purified by size exclusionchromatography. To determine if chemical conjugation of variant IGF2peptide improves rhGAA affinity for the IGF2/CI-MPR receptor, thebinding of unconjugated rhGAA and vIGF2-rhGAA was directly compared atvarying protein concentrations (0.003-10 μg/ml corresponding to 0.012-42nM rhGAA) in receptor plate binding assays FIG. 6A. Significantly higheramounts of captured enzyme activity were observed for vIGF2-rhGAA thanfor unconjugated rhGAA at all protein concentrations tested in theseIGF2/CI-MPR receptor plate binding assays. These results confirm thatconjugation of IGF2 peptide increases rhGAA affinity for the IGF2/CI-MPRreceptor. Moreover, the inclusion of free wildtype IGF2 peptide greatlyreduced vIGF2-rhGAA capture in these plate assays indicating thatbinding was dependent on IGF2 peptide. Much higher amounts of freewildtype IGF2 peptide is likely required to completely eliminatevIGF2-rhGAA binding in these receptor plate assays. To determine whetherincreased receptor affinity would lead to improved cellular uptake forvIGF2-rhGAA, the internalization of extracellular unconjugated rhGAA andvIGF2-rhGAA was evaluated in L6 rat skeletal muscle myoblasts FIG. 6B.vIGF2-rhGAA was shown to be internalized substantially better thanunconjugated rhGAA in L6 myoblasts at all protein concentrations tested.These results demonstrate the functional benefit of improving receptorbinding affinity for enhancing internalization and delivery of exogenouslysosomal enzymes in target cells.

FIGS. 7A and 7B show characterization of variant IGF2 peptide-conjugatedI2S. Variant IGF2 peptide was modified with the crosslinkerNHS-PEG4-azide and subsequently coupled to phosphine-modified I2S. Theresultant variant IGF2 peptide-conjugated I2S (designated as vIGF2-I2S)was purified by size exclusion chromatography. To determine if chemicalconjugation of variant IGF2 peptide improves I2S affinity for theIGF2/CI-MPR receptor, the binding of unconjugated I2S and vIGF2-I2S wasdirectly compared at varying protein concentrations (0.03-10 μg/ml) inreceptor plate binding assays FIG. 7A. Substantially higher amounts ofvIGF2-I2S were captured in these IGF2/CI-MPR receptor plate bindingassays than unconjugated I2S at all protein concentrations tested. Thesereceptor binding results are consistent with those for vIGF2-rhGAA andshow that the same variant IGF2 peptide can be chemically coupled todifferent lysosomal enzymes to increase their binding affinity for theIGF2/CI-MPR receptor. To determine whether multiple variant IGF2peptides can be chemically conjugated to lysosomal enzymes, themolecular mass of unconjugated I2S and vIGF2-I2S was compared by sodiumdodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) FIG. 7B.Unconjugated I2S had an apparent molecular weight of approximately 80kDa (lane 1) on SDS-PAGE while vIGF2-I2S had a much higher apparentmolecular weight of approximately 120 kDa (lane 2). These data indicatethat multiple variant IGF2 peptides must have been chemically conjugatedonto I2S for an increase of approximately 40 kDa since the molecularmass for variant IGF2 peptide is only ˜8 kDa (lane 3). These resultsalso show that I2S was completely converted to vIGF2-I2S with varyingamounts of variant IGF2 peptides as evidenced by the broad protein bandon SDS-PAGE.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present subject matter may be understood more readily by referenceto the following detailed description taken in connection with theaccompanying figures and examples, which form a part of this disclosure.It is to be understood that this invention is not limited to thespecific devices, methods, applications, conditions or parametersdescribed and/or shown herein, and that the terminology used herein isfor the purpose of describing particular embodiments by way of exampleonly and is not intended to be limiting of the claimed invention.

Also, as used in the specification including the appended claims, thesingular forms “a,” “an,” and “the” include the plural, and reference toa particular numerical value includes at least that particular value,unless the context clearly dictates otherwise. The term “plurality”, asused herein, means more than one. When a range of values is expressed,another embodiment includes from the one particular value and/or to theother particular value. Similarly, when values are expressed asapproximations, by use of the antecedent “about,” it is understood thatthe particular value forms another embodiment. All ranges are inclusiveand combinable.

Examples are provided to assist in a further understanding of theinventions. Particular materials used, protocols and conditions areintended to be further illustrative of the inventions and should not beconstrued to limit the reasonable scope thereof.

Suitable methods for conjugating a targeting peptide to a recombinantlysosomal enzyme include modifying the amino (N)-terminus and one ormore lysine residues on a recombinant human lysosomal enzyme using afirst crosslinking agent to give rise to a first crosslinking agentmodified recombinant human lysosomal enzyme, modifying the amino(N)-terminus of a short extension linker region preceding a variantIGF-2 peptide using a second crosslinking agent to give rise to a secondcrosslinking agent modified variant IGF-2 peptide, and then conjugatingthe first crosslinking agent modified recombinant human lysosomal enzymeto the second crosslinking agent modified variant IGF-2 peptidecontaining a short extension linker.

Other suitable methods of conjugating a targeting peptide to arecombinant lysosomal enzyme include conjugating a first crosslinkingagent modified recombinant human lysosomal enzyme to one or more secondcrosslinking agent modified variant IGF-2 peptides, wherein the firstcrosslinking agent modified recombinant lysosomal enzyme comprises arecombinant lysosomal enzyme characterized as having a chemicallymodified N-terminus and one or more modified lysine residues and the oneor more second crosslinking agent modified variant IGF-2 peptidescomprise one or more variant IGF-2 peptides comprising a modifiedN-terminal amino acid of a short extension linker preceding IGF2peptide.

Suitable short extension linkers can be 5 to 20 amino acid residues inlength. The short extension linker can also be about 10 amino acids inlength. Suitable short extension linkers can be represented by the aminoacid sequence in SEQ ID NO:3. Other suitable short extension linkers canbe provided using a 5-amino acid flexible GS extension linker(glycine-glycine-glycine-glycine-serine), a 10-amino acid extensionlinker comprising 2 flexible GS linkers, a 15-amino acid extensionlinker comprising 3 flexible GS linkers, a 20-amino acid extensionlinker comprising 4 flexible GS linkers, or any combination thereof.

Suitable methods of making a targeting peptide conjugated to arecombinant lysosomal enzyme wherein the first crosslinking agentmodified recombinant lysosomal enzyme include using a recombinant humanlysosomal enzyme characterized as having a chemically modifiedN-terminus and one or more modified lysine residues that are modifiedusing a first crosslinking agent. Suitable recombinant human lysosomalenzymes include human acid α-glucosidase (rhGAA), human acidα-galactosidase A (GLA), human acid β-glucuronidase (GUS), human acidα-iduronidase A (IduA), human acid iduronidate 2-sulfatase (I2S), humanβ-hexosaminidase A (HexA), human β-hexosaminidase B (HexB), human acidα-mannosidase A, human β-glucocerebrosidase (GlcCerase), human acidlipase (LP A), and any combinations thereof. One or more lysine residuescan also be modified on the recombinant human lysosomal enzyme. Suitablefirst crosslinking agents include succinimidyl 6-hydrazinonicotinateacetone (S-Hynic), sulfo-succinimidyl 6-hydrazinonicotinate acetone(sulfo-S-HyNic), or C6-succinimidyl 6-hydrazino-nicotinamide(C6-S-Hynic), or succinimidyl 4-hydrazidoterephthalate hydrochloride(SHTH), or succinimidyl 4-hydrazinium nicotinate hydrochloride (SHNH) orany combination thereof to introduce hydrazide moieties on lysosomalenzymes for chemical coupling to targeting peptides that containreactive aldehyde groups. Alternatively, lysosomal enzymes can bemodified with N-hydroxysuccinimide ester-phosphine (NHS-phosphine),sulfo-NHS-phosphine, N-hydroxysuccinimide ester-tetraoxapentadecaneacetylene (NHS-PEG4-acetylene) other NHS-(PEG)n-acetyleneheterobifunctional crosslinkers where “n” can range from 3 to 24discrete PEG units, or cleavable heterobifunctional crosslinkers such asNHS-PEG3-S-S-acetylene, or heterobifunctional crosslinkers containingcyclooctynes such as difluorocyclooctyne (DIFO) and dibenzocyclooctyne(DIBO) or any combination thereof for coupling chemically modifiedlysosomal enzymes to chemically modified targeting peptides containingreactive azide groups. Suitable second crosslinking agents formodification of targeting peptides includePEG4-pentafluorobezene-4-formylbenzoate (PEG4-PFB), or succinimidyl4-formylbenzoate (SFB), or C6-succinimidyl 4-formylbenzoate (C6-SFB) tointroduce reactive aldehyde groups onto targeting peptides forconjugation to lysosomal enzymes containing reactive hydrazide groups.Targeting peptides can also be modified with heterobifunctionalcrosslinkers such as N-hydroxysuccinimide ester-azide (NHS-azide) or,N-hydroxysuccinimide ester-tetraoxapentadecane-azide (NHS-PEG4-azide) orother NHS-(PEG)n-azide crosslinkers where n can range from 3 to 24discrete PEG units, or cleavable heterobifunctional crosslinkers such asNHS-PEG3-S-S-azide, or any combination thereof to introduce reactiveazide groups onto targeting peptides for conjugation to lysosomalenzymes containing reactive phosphines, or alkynes or cyclooctynesgroups. In a preferred embodiment, the first crosslinking agent can beN-succinimidyl 6-hydrazinonicotinate acetone (S-Hynic) and the secondcrosslinking agent can be PEG4-pentafluorobezene-4-formylbenzoate(PEG4-PFB).

The N-terminus and one or more lysine residues on the recombinant humanlysosomal enzyme can be modified in a buffer in the absence of primaryamines at about pH 7.3 at about room temperature for about 30 minutes.The recombinant human lysosomal enzyme can be quickly exchanged into anacidic buffer after the N-terminus and lysine residues on therecombinant human lysosomal enzyme are modified. For example, the acidicbuffer can be 50 mM sodium acetate, at about pH 5.0. The acidic buffercan be 0.1M sodium acetate, potassium acetate, sodium citrate, MES,sodium phosphate or potassium phosphate at about pH 5.0. The exchangeinto an acidic buffer can be carried out using size exclusionchromatography, and the exchange into an acidic buffer can be carriedout using dialysis.

The second crosslinking agent modified variant IGF-2 peptide containinga short linker can be purified before conjugation to the firstcrosslinking agent modified recombinant human lysosomal enzyme. Thepurification can be carried out using gel filtration, dialysis orreverse phase chromatography.

The conjugation of hydrazide-modified recombinant human lysosomal enzymeto aldehyde-modified variant IGF-2 peptide containing a short linker canbe carried out in acidic buffer at about pH 5.0 in the presence ofaniline. The conjugation of phosphine-or acetylene- orcyclooctyne-modified recombinant human lysosomal enzyme toazide-modified variant IGF-2 peptide containing a short linker can becarried out in buffers ranging between pH 5.0-7.0. Recombinant humanlysosomal enzyme-modified IGF-2 peptide containing a short linkerconjugate can be purified using size exclusion chromatography ordialysis.

A suitable first crosslinking agent includes succinimidyl6-hydrazinonicotinate acetone (S-Hynic), sulfo-succinimidyl6-hydrazinonicotinate acetone (sulfo-S-HyNic), or C6-succinimidyl6-hydrazino-nicotinamide (C6-S-Hynic), or succinimidyl4-hydrazidoterephthalate hydrochloride (SHTH), or succinimidyl4-hydrazinium nicotinate hydrochloride (SHNH) or any combination thereofto introduce hydrazide moieties on lysosomal enzymes for chemicalcoupling to targeting peptides that contain reactive aldehyde groups.Alternatively, lysosomal enzymes can be modified withN-hydroxysuccinimide ester-phosphine (NHS-phosphine),sulfo-NHS-phosphine, N-hydroxysuccinimide ester-tetraoxapentadecaneacetylene (NHS-PEG4-acetylene) other NHS-(PEG)n-acetyleneheterobifunctional crosslinkers where “n” can range from 3 to 24discrete PEG units, or cleavable heterobifunctional crosslinkers such asNHS-PEG3-S-S-acetylene, or heterobifunctional crosslinkers containingcyclooctynes such as difluorocyclooctyne (DIFO) and dibenzocyclooctyne(DIBO) or any combination thereof for coupling these chemically modifiedlysosomal enzymes to targeting peptides that contain reactive azidegroups. Suitable second crosslinking agents for modifying targetingpeptides include PEG4-pentafluorobezene-4-formylbenzoate (PEG4-PFB), orsuccinimidyl 4-formylbenzoate (SFB), or C6-succinimidyl 4-formylbenzoate(C6-SFB), or N-hydroxysuccinimide ester-tetraoxapentadecane-azide(NHS-PEG4-azide), or other NHS-(PEG)n-azide heterobifunctionalcrosslinkers where “n” can range from 3 to 24 discrete PEG units, orcleavable heterobifunctional crosslinkers such as NHS-PEG3-S-S-azide. Inanother suitable embodiment, the first crosslinking agent can beN-hydroxysuccinimide ester-phosphine (NHS-phosphine) orsulfo-NHS-phosphine and the second crosslinking agent can beN-hydroxysuccinimide ester-tetraoxapentadecane-azide (NHS-PEG4-azide).In another suitable embodiment, the first crosslinking agent can beN-hydroxysuccinimide ester-tetraoxapentadecane acetylene(NHS-PEG4-acetylene) or other NHS-(PEG)n-acetylene heterobifunctionalcrosslinkers where “n” can range from 3 to 24 PEG units, or cleavableheterobifunctional crosslinkers such as NHS-PEG3-S-S-acetylene and thesecond crosslinking agent can be N-hydroxysuccinimideester-tetraoxapentadecane-azide (NHS-PEG4-azide). In another suitableembodiment, the first crosslinking can be cyclooctynes such asdifluorocyclooctyne (DIFO) and dibenzocyclooctyne (DIBO) and the secondcrosslinking agent can be N-hydroxysuccinimideester-tetraoxapentadecane-azide (NHS-PEG4-azide).

The N-terminus and one or more lysine residues on the recombinant humanlysosomal enzyme can be modified in a buffer lacking primary amines atabout pH 7.3 at about room temperature for about 30 minutes. Therecombinant human lysosomal enzyme can be quickly exchanged into anacidic buffer after the N-terminus and lysine residues on therecombinant human lysosomal enzyme are modified. A suitable acidicbuffer includes 50 mM sodium acetate, at about pH 5.0. The acidic buffercan be 0.1M sodium acetate, potassium acetate, sodium citrate, MES,sodium phosphate or potassium phosphate at about pH 5. The exchange intoan acidic buffer can be suitably carried out using size exclusionchromatography or using dialysis.

The second crosslinking agent modified variant IGF-2 peptide containinga short linker before can be purified prior to conjugation to the firstcrosslinking agent modified recombinant human lysosomal enzyme using gelfiltration, dialysis or reverse phase chromatography. The conjugation ofhydrazide-modified recombinant human lysosomal enzyme toaldehyde-modified variant IGF-2 peptide containing a short linker can becarried out in acidic buffer at about pH 5.0 in the presence of aniline.The conjugation of phosphine- or acetylene- or cyclooctyne-modifiedrecombinant human lysosomal enzyme to azide-modified variant IGF-2peptide containing a short linker can be carried out in buffers rangingbetween pH 5.0-7.0. Recombinant human lysosomal enzyme-modified IGF-2peptide containing a short linker conjugate can be purified using sizeexclusion chromatography or dialysis.

After conjugation, the recombinant human lysosomal enzyme-variant IGF-2peptide containing a short linker can be purified using size exclusionchromatography or dialysis.

The conjugation of the first crosslinking agent (HS-PEG4-acetylene)modified recombinant human lysosomal enzyme to the second crosslinkingagent (HS-PEG4-azide) modified variant IGF-2 peptide containing a shortlinker in acidic buffer at about pH 5.0 can be carried out in thepresence of copper (Cu⁺¹). Following this conjugation step, apurification step of the recombinant human lysosomal enzyme-modifiedIGF-2 peptide containing a short linker conjugate can be carried outusing size exclusion chromatography or dialysis.

The conjugation of the first crosslinking agent (cyclooctyne such asdifluorocyclooctyne; DIFO) modified recombinant human lysosomal enzymeto the second crosslinking agent (HS-PEG4-azide) modified variant IGF-2peptide containing a short linker in acidic buffer at about pH 6.0.Following this conjugation step, a purification step of the recombinanthuman lysosomal enzyme-modified IGF-2 peptide containing a short linkerconjugate can be carried out using size exclusion chromatography ordialysis.

Molecules for enzyme replacement therapy can be generated by conjugatinga heterobifunctional crosslinking agent to a variant IGF-2 peptide andthen conjugating the heterobifunctional crosslinking agent modifiedvariant IGF-2 peptide to a recombinant human lysosomal enzyme. Moleculefor enzyme replacement therapy can also be made by conjugating aheterobifunctional crosslinking agent to a recombinant human lysosomalenzyme and then conjugating the heterobifunctional crosslinking agentmodified recombinant human lysosomal enzyme to a variant IGF-2 peptide.Suitable recombinant human lysosomal enzymes include human acidα-glucosidase (rhGAA), human acid β-galactosidase A (GLA), human acidβ-glucuronidase (GUS), human acid α-iduronidase A (IduA), human acididuronidate 2-sulfatase (I2S), human β-hexosaminidase A (HexA), humanβ-hexosaminidase B (HexB), human acid α-mannosidase A, humanβ-glucocerebrosidase (GlcCerase), human acid lipase (LPA), or anycombination thereof. Suitable heterobifunctional crosslinking agentsinclude m-maleimidobenzyol-N-hydroxysuccinimide ester (MBS),Sulfo-m-maleimidobenzyol-N-hydroxysuccinimide ester (sulfo-MBS) or anycombination thereof. The variant IGF-2 peptide-recombinant humanlysosomal enzyme conjugate can be optionally purified using gelfiltration or dialysis.

Suitable recombinant human lysosomal enzymes can be made using yeast.The recombinant human lysosomal enzyme made from yeast can be treatedusing endoglycosidase F (EndoF) or endoglycosidase H (EndoH) to removeN-glycans. In another suitable embodiment, treatment usingendoglycosidase F (EndoF) or endoglycosidase H (EndoH) can occur inacidic pH buffer. Suitable acidic pH buffers include 0.1M sodiumacetate, pH 5.0. The reactions can be carried out at about roomtemperature. After treatment using endoglycosidase F (EndoF) orendoglycosidase H (EndoH) the recombinant human lysosomal enzyme canoptionally be purified using size exclusion chromatography or dialysis.

Conjugates of one or more variant IGF-2 peptides chemically linked to arecombinant human lysosomal enzyme are also provided. In theseembodiments, the first crosslinking agent modified recombinant lysosomalenzyme can be a recombinant human lysosomal enzyme, and the recombinanthuman lysosomal enzyme can have one or more modified lysine residues,for example the N-terminus can be chemically modified. Suitable variantIGF-2 peptides can be an IGF-2 peptide analog and a short linker with atthe N-terminus. At least one of the variant IGF-2 peptides is suitablyan IGF-2 peptide that has a modified N-terminus within a short linker. Asuitable modified IGF-2 peptide is characterized as being capable ofbeing modified at the N-terminus in a buffer at about pH 7.5. Suitablevariant IGF-2 peptides include a synthetic IGF-2 peptide analog,containing a short linker at the N- or C-terminus with the appropriatereactive chemical group. Suitable variant IGF-2 peptides comprise anIGF-2 peptide analog, a short linker at the N-terminus can be generatedas a recombinant protein and the N-terminal amino acid can besubsequently chemically modified with bifunctional crosslinkers.

A suitable recombinant human lysosomal enzyme includes a human acidα-glucosidase (rhGAA). Other suitable recombinant human lysosomalenzymes that can be used in these methods include human acidα-galactosidase A (GLA), human acid β-glucuronidase (GUS), human acidα-iduronidase A (IduA), human acid isuronidate 2-sulfatase (I2S), humanβ-hexosaminidase A (HexA), human β-hexosaminidase B (HexB), human acidα-mannosidase A, human β-glucocerebrosidase (GlcCerase), human acidlipase (LPA), or any combination thereof. Suitable recombinant humanlysosomal enzymes are characterized as having a modified N-terminus andat least one modified lysine residue.

Suitable first crosslinking agent modified recombinant lysosomal enzymescan be characterized as having a crosslinking agent derived from anamino-reactive bifunctional crosslinker. A suitable first crosslinkingagent modified recombinant lysosomal enzyme can be characterized ascomprising a crosslinking agent derived from succinimidyl6-hydrazinonicotinate acetone (S-Hynic), sulfo-succinimidyl6-hydrazinonicotinate acetone (sulfo-S-HyNic), or C6-succinimidyl6-hydrazino-nicotinamide (C6-S-Hynic), or succinimidyl4-hydrazidoterephthalate hydrochloride (SHTH), or succinimidyl4-hydrazinium nicotinate hydrochloride (SHNH) or any combination thereofto introduce hydrazide moieties. Alternatively, lysosomal enzymes can bemodified with N-hydroxysuccinimide ester-phosphine (NHS-phosphine),sulfo-NHS-phosphine, N-hydroxysuccinimide ester-tetraoxapentadecaneacetylene (HS-PEG4-acetylene) other NHS-(PEG)n-acetyleneheterobifunctional crosslinkers where “n” can range from 3 to 24discrete PEG units, or cleavable heterobifunctional crosslinkers such asNHS-PEG3-S-S-acetylene, or heterobifunctional crosslinkers containingcyclooctynes such as difluorocyclooctyne (DIFO) and dibenzocyclooctyne(DIBO) or any combination thereof for coupling these chemically modifiedlysosomal enzymes to targeting peptides that contain reactive azidegroups. The modified N-terminus and lysine residues on the recombinanthuman lysosomal enzyme can be characterized as being derived from theprimary amine on the first (N-terminal) amino acid and lysine residuesmodified in a buffer lacking primary amines at about pH 7.3 at aboutroom temperature for about 30 minutes. Variant IGF-2 peptides can alsoinclude the IGF-2 peptide and a short extension linker coupled to asecond crosslinking agent. A suitable second crosslinking agent can bePEG4-pentafluorobezene-4-formylbenzoate (PEG4-PFB) for conjugation tosuccinimidyl 6-hydrazinonicotinate acetone (S-Hynic)-modified lysosomalenzymes. In a different embodiment, the second crosslinking agent cancomprise NHS-PEG4-azide for conjugation to phosphine-modified lysosomalenzymes. In another embodiment, the second crosslinking agent cancomprise N-hydroxysuccinimide ester-PEG4-azide (NHS-PEG4-azide) forconjugation to acetylene-modified lysosomal enzymes. In yet anotherembodiment, the second crosslinker can comprise N-hydroxysuccinimideester-PEG4-azide (NHS-PEG4-azide) for conjugation tocyclooctyne-modified lysosomal enzyme.

A heterobifunctional crosslinking agent modified variant IGF-2 peptideconjugated to a recombinant human lysosomal enzyme is also provided.Suitable heterobifunctional crosslinking agent modified variant IGF-2peptides are characterized as being derived from a heterobifunctionalcrosslinking agent conjugated to a variant IGF-2 peptide. A suitablerecombinant human lysosomal enzyme can be human acid α-glucosidase(rhGAA), human acid α-galactosidase A (GLA), human acid β-glucuronidase(GUS), human acid α-iduronidase A (IduA), human acid iduronidate2-sulfatase (I2S), human β-hexosaminidase A (HexA), humanβ-hexosaminidase B (HexB), human acid α-mannosidase A, humanβ-glucocerebrosidase (GlcCerase), human acid lipase (LPA). A suitableheterobifunctional crosslinking agent includesm-maleimidobenzyol-N-hydroxysuccinimide ester (MBS andsulfo-m-maleimidobenzyol-N-hydroxysuccinimide ester (sulfo-MBS),Sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate(sulfo-SMCC). The conjugates can be substantially pure with less than 10percent of free, unconjugated IGF2 peptide. The purity of the conjugatecan be measured by absorbance with lysosomal protein at 280 nm and freeIGF2 peptide at 214 nm in fractions from size exclusion chromatographyor by stained protein gels using sodium docecylsulfate polyacrylamidegel electrophoresis (SDS-PAGE) or by Western blotting after SDS-PAGE andspecific antibodies for detection of lysosomal enzymes or IGF2 peptide.The conjugate can also be substantially pure with less than 0.1 percentof free, unconjugated IGF2 peptide or other contaminants. Therecombinant human lysosomal enzyme can be suitably derived from yeastwith high-mannose or complex-type N-glycans. Suitable recombinant humanlysosomal enzymes derived from yeast with complex-type N-glycans can beused directly for conjugation to IGF2 peptide. Suitable recombinanthuman lysosomal enzymes with high-mannose type N-glycans can also betreated using endoglycosidase F (EndoF) or endoglycosidase H (EndoH) toremove these exotic N-glycans prior to or after chemical conjugation.The recombinant human lysosomal enzyme can be suitably derived fromother protein expression systems including insect cells, plant cells,fungi, transgenic animals and in vitro translation systems.

Methods for treating a subject suffering from a lysosomal storagedisease are carried out by administering to the subject a conjugate ofone or more variant IGF-2 peptides chemically conjugated to a chemicallymodified recombinant human lysosomal enzyme. Any of a variety oflysosomal storage diseases can be treated this way, including at leastone of the following diseases: Pompe Disease, Fabry Disease, and GaucherDisease, MPS I, MPSII, MPS VII, Tay Sachs, Sandhoff, α-mannosidosis, andWohlman.

Methods for treating a subject suffering from a lysosomal storagedisease are carried out by administering to the subject a conjugate of aheterobifunctional crosslinking agent modified variant IGF-2 peptideconjugated to a recombinant human lysosomal enzyme. Any of a variety oflysosomal storage diseases can be treated this way, including at leastone of the following diseases: Pompe Disease, Fabry Disease, and GaucherDisease, MPS I, MPSII, MPS VII, Tay Sachs, Sandhoff, α-mannosidosis, andWohlman.

Methods of treating a patient suffering from Pompe, Fabry, Gaucher, MPSI, MPSII, MPS VII, Tay Sachs, Sandhoff, α-mannosidosis, or Wohlmandisease is also provided by administering to a patient in need thereof,a composition of one or more variant IGF-2 peptides chemicallyconjugated to a recombinant lysosomal enzyme and a pharmaceuticallyacceptable carrier, in an amount sufficient to treat the disease. Asuitable modified recombinant human lysosomal enzyme includes acidα-glucosidase for the treatment of Pompe disease. The modifiedrecombinant human lysosomal enzyme can also be acid α-galactosidase Afor the treatment of Fabry disease. The modified recombinant humanlysosomal enzyme can be acid β-glucocerebrosidase for the treatment ofGaucher disease. The modified recombinant human lysosomal enzyme can beacid α-iduronidase for the treatment of mucopolysaccharidosis I (MPS I).The modified recombinant human lysosomal enzyme can be acid iduronidate2-sulfatase for the treatment of mucopolysaccharidosis II (MPS II). Themodified recombinant human lysosomal enzyme can also be acidβ-glucuronidase for the treatment of mucopolysaccharidosis VII (MPSVII). Alternatively, the modified recombinant human lysosomal enzyme canbe β-hexosaminidase A for the treatment of GM2 gangliosidoses(Tay-Sachs). In another suitable embodiment the modified recombinanthuman lysosomal enzyme can be β-hexosaminidase B for the treatment ofGM2 gangliosidoses (Sandhoff). In another embodiment the modifiedrecombinant human lysosomal enzyme can be acid lipase for the treatmentof Wohlman disease. The modified recombinant human lysosomal enzyme canalso be acid α-mannosidase for the treatment of α-mannosidosis. Thecompositions provided herein can be administered in an amount of fromabout 0.1 to about (100 milligrams of one or more variant IGF-2 peptideschemically conjugated to a recombinant lysosomal enzyme per patientkilogram per month. In another suitable embodiment the composition canbe administered in an amount of from about 1 to about 500 milligrams ofone or more variant IGF-2 peptides chemically conjugated to arecombinant lysosomal enzyme per patient per kilogram per month.

Methods of treating a patient suffering from Pompe, Fabry, Gaucher, MPSI, MPS II, MPS VII, Tay Sachs, Sandhoff, α-mannosidosis, Wohlman diseaseare also provided by administering to a patient in need thereof, acomposition of a heterobifunctional crosslinking agent modified variantIGF-2 peptide conjugated to a recombinant human lysosomal enzyme and apharmaceutically acceptable carrier, in an amount sufficient to treatthe disease. The composition can be administered in an amount of fromabout 0.1 to about 1000 milligrams of a heterobifunctional crosslinkingagent modified variant IGF-2 peptide conjugated to a recombinant humanlysosomal enzyme per 50 kilograms of patient per month. In anothersuitable embodiment, the composition can be administered in an amount offrom about 1 to about 500 milligrams of a heterobifunctionalcrosslinking agent modified variant IGF-2 peptide conjugated to arecombinant human lysosomal enzyme per 50 kilograms of patient permonth.

A suitable DNA sequence that encodes a variant IGF-2 peptide that isoptimized for expression in E. coli is provided as SEQ ID NO: 1. Asuitable amino acid sequence that represents a variant IGF-2 peptide isprovided as SEQ ID NO: 2. A suitable amino acid sequence that representsan extension linker is provided as SEQ ID NO: 3. The variant IGF2peptide used in the methods can have the amino acid sequence of SEQ IDNO: 2. In another embodiment the variant IGF2 peptide in the conjugatescan have the amino acid sequence of SEQ ID NO: 2.

EXAMPLES AND OTHER ILLUSTRATIVE EMBODIMENTS

A chemical crosslinking method is employed to conjugate variant humanIGF-2 peptides to lysosomal enzymes for developing novel and superiorERTs for the treatment of various lysosomal storage disorders (LSDs).This strategy is expected to increase the binding affinity of IGF2peptide-conjugated ERTs for the IGF-2/CI-MPR and improve cellular uptakeand delivery of these recombinant enzymes to lysosomes. By doing so,these IGF2 peptide-conjugated ERTs are expected to be more effective inclearing accumulated substrate in affected cells.

Several different variants of human IGF-2 peptides can be synthesized orexpressed (in mammalian cells or in other organisms), purified andsubsequently chemically modified with heterobifunctional crosslinkersfor conjugation to lysosomal enzymes. A variant IGF-2 peptide cancontain one or combinations of following modifications: substitution ofarginine for glutamic acid at position 6; deletion of amino acids 1-4and 6; deletion of amino acids 1-4 and 6, 7; deletion of amino acids 1-4and 6 and substitution of lysine for threonine at position 7; deletionof amino acids 1-4 and substitution of glycine for glutamic acid atposition 6 and substitution of lysine for threonine at position 7;substitution of leucine for tyrosine at position 27; substitution ofleucine for valine at position 43; substitution of arginine for lysineat position 65. The majority of these modifications are designed toreduce binding affinity of IGF-2 peptides for the insulin and IGF-1receptors and for serum IGF binding proteins (IGFBPs) while maintaininghigh affinity for the IGF-2/CI-MPR. The modified IGF-2 peptides may alsocontain an affinity tag (e.g., polyhistidine; His tag) for rapidpurification of the modified IGF-2 peptide, may be expressed as fusionproteins with soluble protein partners, a protease site (e.g., enhancedtobacco etch virus (TEV) protease site) for removal of the affinity tagor fusion protein partner, a linker extension region of at least fiveamino acids preceding IGF-2.

Variant IGF-2 peptides and recombinant lysosomal enzymes can bechemically coupled by two primary strategies. (A) Independently modifythe IGF-2 peptide with a heterobifunctional crosslinker and therecombinant lysosomal enzyme with a different heterobifunctionalcrosslinker (as described in examples 1-3). After purification to removeexcess, unconjugated crosslinker and chemical byproducts, thechemically-modified IGF2 peptide and chemically-modified lysosomalenzyme are subsequently conjugated together in a final chemical reactionto form the IGF2 peptide-lysosomal enzyme conjugate and purified andstored in an acidic pH buffer to maintain enzyme activity. (B)Chemically conjugate the IGF2 peptide and lysosomal enzyme using asingle heterobifunctional crosslinker (as described in example 4). TheIGF-2 peptide is chemically modified with the heterobifunctionalcrosslinker at one pH reaction condition. The chemically modifiedlysosomal enzyme is then added and the pH adjusted to a second pHreaction condition to conjugate the IGF2 peptide to lysosomal enzyme.The conjugate is then be purified to remove excess, unconjugatedheterobifunctional crosslinker and chemical byproducts and stored in anacidic pH buffer to maintain enzyme activity.

The above chemical coupling approach has distinct advantages forimproving protein targeting for lysosomal enzyme replacement therapies.First, conjugation of modified IGF-2 peptides increases binding affinityof lysosomal enzymes for the IGF-2/CI-MPR without requiring specializedM6P carbohydrate structures. Second, unlike IGF-2 fusion proteins whichcontains a single IGF-2 peptide per lysosomal enzyme, this strategy canappend multiple modified IGF-2 peptides to lysosomal enzymes for higheraffinity for the IGF-2/CI-MPR. Third, this approach can be used toconjugate mixed peptides (IGF2 peptide and other peptides) for improvingdrug targeting to other tissues (e.g., the brain). Fourth, this approachcan utilize recombinant lysosomal enzymes produced from most eukaryoticexpression systems including but not limited to mammalian cells, yeast,insect cells, plant cells, transgenic animals (e.g., in hen eggs, milk,etc.). Recombinant lysosomal enzymes that contain complex-type N-glycans(i.e., derived from mammalian expression systems, yeast with modifiedN-glycan processing that yield complex N-glycans, transgenic animals,etc.) can be directly utilized for coupling. Enzymes bearinghigh-mannose type N-glycans (i.e., derived from yeast, Lec1 mammaliancell lines, etc.) can be subjected to deglycosylation (viaendoglycosidases such as EndoF or EndoH) prior to or after chemicalcoupling to modified IGF-2 peptides (as described in example 5). Fifth,modified IGF-2 peptides can be manufactured in most expression systemsincluding bacteria, yeast or other fungal systems which enable acost-effective approach for scale up of process. Sixth, the samemodified IGF-2 peptides can be conjugated to any lysosomal enzyme toimprove protein targeting without having to create individual fusionproteins of IGF2-lysosomal enzyme. Seventh, this strategy can createnovel, superior ERT compositions that potentially could reduce drugrequirements, decrease infusion time and reduce immunogenicity.

Example 1

Recombinant human acid α-glucosidase (rhGAA) derived from most mammaliancell manufacturing systems contain very low amounts of M6P with mostlycomplex-type N-glycans that are not adequate for high affinity bindingof rhGAA to the IGF-2/CI-MPR. This N-glycan profile resembles that forserum proteins and thus, enables rhGAA to have a favorablepharmacokinetic profile (i.e., slower clearance) in the circulation.rhGAA can therefore be utilized for conjugation to modified IGF-2peptides to increase its affinity for the IGF-2/CI-MPR for improvedprotein targeting and cellular uptake to develop a superior rhGAA ERT.Specifically, rhGAA can be concentrated to a protein concentration of8-10 mg/ml and exchanged into buffers at about pH 7.3 lacking primaryamines (e.g., 50 mM sodium phosphate, pH 7.3/100 mM NaCl) andsubsequently modified with a 12- to 20-fold molar excess of theheterobifunctional crosslinker succinimidyl 6-hydrazinonicotinateacetone (S-Hynic) at room temperature for about 30 min. In thisreaction, the chemically reactive N-hydroxysuccinimide ester (NHS) groupfrom S-Hynic reacts with the α-amino group of the first amino acidresidue at the amino (N)-terminus and ε-amino groups of lysines on rhGAAto introduce novel, chemically active hydrazide groups at these modifiedamino acid residues. The S-Hynic-modified rhGAA then quickly exchangesinto acidic buffer (e.g., 50 mM NaOAc, pH 4.8/100 mM NaCl/0.05%Polysorbate-80) via size exclusion chromatography or dialysis to removeexcess crosslinker and chemical byproducts and to preserve enzymaticactivity. This chemical reaction can be titrated with varying amounts ofS-Hynic (e.g., 5-40× molar excess) to understand the ratio of S-Hynic torhGAA that reproducibly yields 1-4 chemically-active hydrazide groups onrhGAA. The optimal conditions are then used for scaling up the S-Hynicmodification reaction of rhGAA.

In a separate reaction, a variant IGF2 peptide such as [del(1-4), Arg6,Leu27, Arg65] IGF-2 containing a short extension linker region (atN-terminus), is chemically modified using the heterobifunctionalcrosslinker PEG4-pentafluorobezene-4-formylbenzoate (PEG4-PFB) atpH˜7.5, room temperature for 2-3 hours. In this reaction, the PEG4-PFBmodifies the α-amino group of the first amino acid glycine from theshort extension linker region to introduce a novel reactive aldehydechemical group at the amino terminus. The chemical modification ofvariant IGF2 peptide can be monitored by C4 reverse phase chromatographyto assess the progression and completeness of chemical modification asshown in FIGS. 5A-5C. The PEG4-benzaldehyde-modified IGF-2 peptide isthen purified by gel filtration chromatography or dialysis to removeexcess crosslinker and chemical byproducts in an appropriate buffer forconjugation (e.g., 50 mM NaOAc, pH 4.8/100 mM NaCl/0.05%Polysorbate-80). A final reaction is then performed to conjugate theS-Hynic-modified rhGAA to the PEG4-benzaldehyde-modified IGF-2 peptidein 50 mM NaOAc, pH 4.8/100 mM NaCl/0.05% Polysorbate-80 buffer over a 24hr period at room temp. This chemistry couples the hydrazide groups fromthe S-Hynic-modified rhGAA to chemically-active aldehyde groups fromPEG4-benzaldehyde-modified IGF2 peptides to form stable covalent(hydrazone) bonds. This reaction can be performed in the presence ofaniline (e.g., 10 mM) with varying amounts of PEG4-benzaldehyde-modifiedIGF-2 peptide (e.g., 1-10× molar excess) to optimize coupling. The IGF-2peptide-conjugated rhGAA is then purified by size exclusionchromatography or dialysis against 50 mM sodium phosphate, pH 6.2/100 mMNaCl/0.05% Polysorbate-80 to remove excess PEG4-benzaldehyde-modifiedIGF-2 peptides and the variant IGF2 peptide-conjugated rhGAA(vIGF2-rhGAA) is stored in the same buffer at 4° C. or frozen at −20° C.or −70° C.

Example 2

Recombinant human acid α-glucosidase (rhGAA) derived from mammalianmanufacturing systems are utilized for conjugation to variant IGF-2peptides to increase affinity for the IGF-2/CI-MPR for improved proteintargeting and cellular uptake to develop a superior rhGAA ERT.Specifically, the Staudinger Ligation (azide-phosphine) reactionchemistry is used to couple IGF2 peptides to rhGAA to generate an IGF2peptide-rhGAA conjugate for improved drug targeting. In this example,rhGAA (at 5-10 mg/ml) is exchanged into buffers at about pH 7.3 lackingprimary amines (e.g., 50 mM sodium phosphate (pH 7.3)/00) mM NaCl) andsubsequently is modified with 10- to 20-fold molar excess of theheterobifunctional crosslinker sulfo-N-hydroxysuccinimideester-phosphine (sulfo-NHS-phosphine) at room temperature for about 30min. In this chemical reaction, the chemically reactive NHS group fromsulfo-NHS-phosphine reacts with the α-amino group of the first aminoacid residue at the N-terminus and ε-amino groups of lysines on rhGAA tointroduce novel, chemically active phosphine groups at these modifiedamino acid residues. The phosphine-containing rhGAA is then quicklyexchanged into slightly acidic buffer (e.g., 50 mM sodium phosphate, pH6.5/100 mM NaCl) via size exclusion chromatography or dialysis to removeexcess crosslinker and chemical byproducts and to preserve enzymaticactivity. This chemical reaction can be titrated with varying amounts ofsulfo-NHS-phosphine (e.g., 5-40× molar excess) to understand the ratioof sulfo-NHS-phosphine to rhGAA that reproducibly yields 1-4chemically-active phosphine groups on rhGAA. The optimal conditions canbe used for scaling up the sulfo-NHS-phosphine modification reaction ofrhGAA.

In a separate reaction, a variant IGF-2 peptide such as [del(1-4), Arg6,Leu27, Arg65] IGF-2 containing a short extension linker region (atN-terminus), is chemically modified using a 30-fold molar excess of theheterobifunctional crosslinker N-hydroxysuccinimide ester-PEG4-azide(NHS-PEG4-azide) in a pH˜7.5 buffer lacking primary amines (e.g., 50 mMsodium phosphate/50 mMNaCl, pH 7.5) at room temp for 1-3 hrs. In thisreaction, the reactive NHS group of NHS-PEG4-azide is reacted with theα-amino group of glycine from the short extension linker region tointroduce a novel azide chemical group at the N-terminus. The chemicalmodification of variant IGF2 peptide can be monitored by C4 reversephase chromatography to assess the progression and completeness ofchemical modification. The PEG4-azide-modified IGF-2 peptide is thenpurified by C4 reverse phase chromatography and the modified peptide islyophilized to remove solvents and stored as a dry powder.

A final reaction is then performed to conjugate the phosphine-modifiedrhGAA to the PEG4-azide-modified IGF-2 peptide by directly addingphosphine-modified rhGAA (in 50 mM sodium phosphate, pH 6.5/100 mM NaClbuffer) to the freeze dried PEG4-azide-modified IGF-2 peptide at a molarratio of 1 part rhGAA to 5 parts IGF2 peptide with incubation at roomtemp over a 24 hr period. This chemistry couples the azide chemicalgroup from the azide-modified IGF-2 peptide to phosphine-modified rhGAAto form stable covalent (amide) bonds. The variant IGF-2peptide-conjugated rhGAA (vIGF2-rhGAA) is then purified by sizeexclusion chromatography or dialysis to remove excessPEG4-azide-modified IGF-2 peptides and stored in slightly acidic pHbuffer (50 mM sodium phosphate, pH 6.5/100 mM NaCl buffer) at 4° C.

Example 3

Recombinant human acid iduronidate 2-sulfatase (I2S) derived frommammalian manufacturing systems is utilized for conjugation to variantIGF-2 peptides to increase enzyme affinity for the IGF-2/CI-MPR forimproved protein targeting and cellular uptake to develop a superior I2SERT. Specifically, the Staudinger Ligation (azide-phosphine) reactionchemistry is used to couple variant IGF2 peptides to I2S to generate anIGF2 peptide-I2S conjugate for improved drug targeting. In this example,I2 S (at approximately 3 mg/ml) is modified with 20-fold molar excess ofthe heterobifunctional crosslinker sulfo-N-hydroxysuccinimideester-phosphine (sulfo-NHS-phosphine) in a pH˜7.3 buffer lacking primaryamines (e.g., 50 mMM sodium phosphate/100 mM NaCl, pH 7.3) at roomtemperature for about 30 min. In this chemical reaction, the chemicallyreactive NHS group from sulfo-NHS-phosphine reacts with the α-aminogroup of the first amino acid residue at the N-terminus and ε-aminogroups of lysines on I2S to introduce novel, chemically active phosphinegroups at these modified amino acid residues. The phosphine-containingI2S is then quickly exchanged into slightly acidic buffer (e.g., 50 mMsodium phosphate, pH 6.5/100 mM NaCl) via size exclusion chromatographyor dialysis to remove excess crosslinker and chemical byproducts and topreserve enzymatic activity. This chemical reaction can be titrated withvarying amounts of sulfo-NHS-phosphine (e.g., 5-40× molar excess) tounderstand the ratio of sulfo-NHS-phosphine to I2S that reproduciblyyields 1-4 chemically-active phosphine groups on I2S. The optimalconditions can be used for scaling up the sulfo-NHS-phosphinemodification reaction of I2S.

In a separate reaction, a variant IGF-2 peptide such as [del(1-4), Arg6.Leu27, Arg65] IGF-2 containing a short extension linker region (atN-terminus), is chemically modified using a 30-fold molar excess of theheterobifunctional crosslinker N-hydroxysuccinimide ester-PEG4-azide(NHS-PEG4-azide) in a pH˜7.5 buffer lacking primary amines (e.g., 50 mMsodium phosphate/50 mMNaCl, pH 7.5) at room temp for 1-3 hrs. In thisreaction, the reactive NHS group of NHS-PEG4-azide is reacted with theα-amino group of glycine from the short extension linker region tointroduce a novel azide chemical group at the N-terminus. The chemicalmodification of variant IGF2 peptide can be monitored by C4 reversephase chromatography to assess the progression and completeness ofchemical modification. The PEG4-azide-modified IGF-2 peptide is thenpurified by C4 reverse phase chromatography and the peptide islyophilized and stored as a dry powder.

A final reaction is then performed to conjugate the phosphine-modifiedI2 S to the PEG4-azide-modified IGF-2 peptide by directly addingphosphine-modified I2S (in 50 mM sodium phosphate, pH 6.5/100 mM NaClbuffer) to the freeze dried PEG4-azide-modified IGF-2 peptide at a molarratio of 1 part I2 S to 5 parts IGF2 peptide with incubation at roomtemp over a 24 hr period. This chemistry couples the reactive azidechemical group from the azide-modified IGF-2 peptide tophosphine-modified I2S to form stable covalent (amide) bonds. Thevariant IGF-2 peptide-conjugated I2S (vIGF2-I2S) is then purified bysize exclusion chromatography or dialysis to remove excessPEG4-azide-modified IGF-2 peptides and stored in slightly acidic pHbuffer (50 mM sodium phosphate, pH 6.5/100 mM NaCl buffer) at 4° C.

Example 4

Recombinant human acid α-glucosidase (rhGAA) derived from mammalianmanufacturing systems will be utilized for conjugation to modified IGF-2peptides to increase affinity for the IGF-2/CI-MPR for improved proteintargeting and cellular uptake to develop a superior rhGAA ERT. In thisexample, a variant IGF2 peptide such as [del(1-4), Arg6, Leu27, Arg65]IGF-2 containing a short extension linker region with a cysteine residueat the N-terminus is modified with the heterobifunctional crosslinkerm-maleimidobenzyol-N-hydroxysuccinimide ester (MBS) at about pH 6 androom temp for 30-60 min. In this reaction, the chemically reactivemaleimide group from MBS will react with the free sulfhydryl group fromthe N-terminal cysteine while preserving the N-hydroxysuccinimide esterreactive group for coupling to rhGAA. The MBS-modified IGF-2 peptidewill be quickly purified by gel filtration chromatography or dialysis toremove excess MBS. rhGAA is then added for coupling to the MBS-modifiedIGF-2 peptide at room temp in non-amine containing buffer at pH 7.3 for30 min. In this chemical reaction, the chemically reactiveN-hydroxysuccinimide ester group (from MBS-modified IGF-2 peptide)reacts with the α-amino group of the first amino acid residue at theN-terminus and ε-amino groups of lysines on rhGAA to form stablecovalent linkages. This reaction will be titrated using varying amountsof MBS-modified IGF-2 peptide (e.g., 1-20× molar excess) to determinethe molar excess of MBS-modified IGF-2 peptide to couple 1-4 IGF-2peptides on rhGAA. The optimal coupling conditions are then used forscaling up this process. The IGF-2-conjugated rhGAA will be purified bygel filtration chromatography or dialysis to remove excess IGF-2peptides and stored in acidic pH buffer (0.1M sodium citrate, pH 5.5buffer).

Example 5

Recombinant human lysosomal enzymes such as rhGAA with high-mannose typeN-glycan structures (derived from yeast, GNT-1 deficient Led mammaliancells, etc.) can be utilized for conjugation to variant IGF-2 peptidesto increase affinity for the IGF-2/CI-MPR for improved protein targetingand cellular uptake to develop a superior rhGAA ERT. In this example,rhGAA (at 8-10 mg/ml) is exchanged into buffers at about pH 7.3 lackingprimary amines (e.g., 50 mM sodium phosphate (pH 7.3)/100 mM NaCl) andsubsequently modified with a heterobifunctional crosslinker such asN-succinimidyl 6-hydrazinonicotinate acetone (S-Hynic) in a pH˜7.3buffer lacking primary amines (e.g., 50 mM sodium phosphate/0.1 M NaCl,pH 7.3) at room temperature for 30 min. The hydrazide-modified rhGAA isthen quickly exchanged into acidic buffer (e.g., 50 mM NaOAc, pH 4.8/100mM NaCl/0.05% Polysorbate-80) via size exclusion chromatography ordialysis to remove excess crosslinker and chemical byproducts and topreserve enzymatic activity.

In a separate reaction, a variant IGF-2 peptide such as [del(1-4), Arg6,Leu27, Arg65] IGF-2 containing a short extension linker region (atN-terminus), is chemically modified using a 30-fold molar excess of theheterobifunctional crosslinker PEG4-pentafluorobezene-4-formylbenzoate(PEG4-PFB) in a pH˜7.5 buffer lacking primary amines (e.g., 50 mM sodiumphosphate/50 mM NaCl, pH 7.5) at room temp for 1-3 hrs. In thisreaction, PEG4-PFB modifies the α-amino group of glycine from the shortextension linker region to introduce a novel aldehyde chemical group atthe N-terminus. The chemical modification of variant IGF2 peptide can bemonitored by C4 reverse phase chromatography to assess the progressionand completeness of chemical modification. ThePEG4-benzaldehyde-modified IGF-2 peptide is then purified by gelfiltration chromatography or dialysis to remove excess crosslinker andchemical byproducts in an appropriate buffer for conjugation (e.g., 50mM NaOAc, pH 4.8/100 mM NaCl/0.05% Polysorbate-80).

A final reaction is then performed to conjugate the S-Hynic-modifiedrhGAA to the PEG4-benzaldehyde-modified IGF-2 peptide in 50 mM NaOAc, pH4.8/100 mM NaCl/0.05% Polysorbate-80 buffer over a 24 hr period at roomtemp. This chemistry couples the hydrazide groups from theS-Hynic-modified rhGAA to chemically-active aldehyde groups fromPEG4-benzaldehyde-modified IGF2 peptides to form stable covalent(hydrazone) bonds. This reaction can be performed in the presence ofaniline (e.g., 10 mM) with varying amounts of PEG4-benzaldehyde-modifiedIGF-2 peptide (e.g., 1-10× molar excess) to optimize coupling. Thevariant IGF-2 peptide-conjugated rhGAA (vIGF2-rhGAA) is then purified bysize exclusion chromatography or dialysis to remove excessPEG4-azide-modified IGF-2 peptides in acidic pH buffer (50 mM NaOAc, pH4.8/100 mM NaCl/0.05% Polysorbate-80).

The high-mannose type N-glycans on rhGAA is problematic because it isbelieved that these carbohydrates cause the protein to be rapidlycleared from the circulation via macrophage and splenic mannosereceptors. However, high-mannose type N-glycans can be removed fromrhGAA under native (i.e., non-denaturing conditions which preservescatalytic activity) using endoglycosidase F (EndoF) or endoglycosidase H(EndoH) in acidic pH buffer (e.g., 50 mM NaOAc, pH 4.8/100 mM NaCl/0.05%Polysorbate-80) at room temperature. rhGAA has been experimentally shownto remain soluble and is completely active after removal of N-glycans(data not shown). Deglycosylation of rhGAA can be performed after theenzyme is modified with S-Hynic and purified (via size exclusionchromatography or dialysis to remove excess crosslinker). This strategyallows for complete deglycosylation of rhGAA over 1-5 days using EndoFor EndoH without affecting enzyme activity. The deglycosylatedhydrazide-modified rhGAA is then conjugated toPEG4-benzaldehyde-modified IGF-2 peptides. Alternatively, rhGAAdeglycosylation can be performed concurrently during the conjugation ofPEG4-benzaldehyde-modified IGF-2 peptide to hydrazide-modified rhGAAusing high concentrations of EndoF or EndoH. Deglycosylated, IGF2peptide-conjugated rhGAA is then purified by gel filtrationchromatography or dialysis to remove excess phosphine-modified IGF-2peptides and stored in acidic pH buffer (50 mM sodium phosphate, pH6.2/100 mM NaCl/0.05% Polysorbate-80). The ideal method to removehigh-mannose N-glycans from yeast-derived rhGAA would be to co-expressEndoH with the lysosomal enzyme for deglycoylation in vivo prior toprotein purification of rhGAA. This would generate deglycosylated rhGAAwhich can be directly modified and coupled to variant targeting peptideswithout any additional processing.

The above strategies enable the removal of undesirable N-glycans fromrhGAA to prevent allergenic responses and to prevent rapid proteinclearance while significantly improving binding affinity for theIGF-2/CI-MPR for improved protein targeting and cellular uptake of IGF2peptide-conjugated lysosomal enzymes. Importantly, this strategy canutilize lysosomal enzymes produced from non-mammalian systems andrepresent a much more cost-effective approach for developing superiorERTs.

The above examples are designed for increasing the affinity of differentlysosomal enzymes for the IGF-2/CI-MPR via chemical conjugation ofmodified IGF-2 peptides. This strategy improves protein targeting forcurrent and future ERTs to develop superior treatments for LSDs.

Example 6

An IGF2/CI-MPR receptor binding assay was utilized to assess the effectsof chemical conjugation of IGF2 peptide on receptor affinity forlysosomal enzymes rhGAA and I2S. This assay is designed to differentiatelysosomal enzymes with high binding affinity for the IGF2/CI-MPR fromthose with low to moderate binding since unbound lysosomal enzymes arewashed away during processing. Moreover, since varying proteinconcentrations of the lysosomal enzymes are used to assess binding, thisassay can determine the protein concentrations required for bindingreceptor which can be utilized to estimate binding affinity for eachlysosomal enzyme preparation. Specifically, unmodified lysosomal enzymesand IGF2 peptide-conjugated lysosomal enzymes were serially diluted in40 mM HEPES (pH 6.7)/150 mM NaCl/10 mM EDTA and then incubated in96-well ELISA plates which were coated with IGF2/CI-MPR receptor (50 μlper well of receptor at 6 μg/mlin phosphate buffered saline; thenblocked with 2% BSA in phosphate buffered saline) for 1 hr at 30° C. Theplates were subsequently washed three times with the same buffercontaining 0.1% Tween-20 to remove unbound proteins. The bound lysosomalenzymes were then measured by enzyme activity using the appropriatefluorogenic substrates (e.g., 4-methylumbelliferyl-α-D-glucopyranoside(4-MU-α-Glc) for rhGAA) in assay buffer (50 mM NaOAc, pH 4.8/2%BSA/0.02% Triton X-100) at 37° C. for 1 hr. The samples were thentransferred to new 96-well plates, 0.1M NaOH was added to raise the pHof solution to approximately 10.5 and the plates were read in afluorescence plate reader at the appropriate excitation and emissionwavelengths (i.e., 370 nm excitation & 460 nm emission for 4-MU). Ourresults show that much higher amounts of bound enzyme activity wereobserved for vIGF2-rhGAA than unconjugated rhGAA at all proteinconcentrations tested as shown in FIG. 6A. The binding of vIGF2-rhGAA toIGF2/CI-MPR plates was reduced significantly by the inclusion of free WThuman IGF2 peptide indicating that this binding was dependent on IGF2peptide. Much higher amounts of free WT human IGF2 peptide is likelyrequired for complete blockade of vIGF2-rhGAA to IGF2/CI-MPR. Chemicalconjugation of IGF2 peptide onto I2 S was also shown substantiallyincrease binding affinity of that enzyme for the IGF2/CI-MPR (FIG. 7A).Moreover, similar amounts of bound I2S activities were observed at 1 and3 μg/ml for the IGF2 peptide-conjugated I2S (vIGF2-I2S) which indicatesthat receptor binding was saturated.

These collective data uncovered several important features of thevariant IGF2 peptide-conjugation approach. (1) The protein structure ofvariant IGF2 peptide is appropriate for high affinity binding toIGF2/CI-MPR receptor. This functional assessment is consistent with ourC4 reverse phase chromatography data that show wildtype and variant IGF2peptides bind and elute at nearly identical conditions as shown in FIGS.5A-5C. Since the “fingerprints” of these two IGF2 peptides are virtuallyindistinguishable on C4 reverse phase chromatography, they must be verysimilar in their protein conformations. (2) The chemical conjugation ofvariant IGF2 peptide did not affect enzyme activity for either rhGAA orI2S (data not shown). The utilization of an extension linker region forchemical coupling of the peptide to lysosomal enzymes likely provided atether that is sufficient for IGF2 peptide binding while maintainingenzyme activity. (3) The conjugated variant IGF2 peptide is stable andmaintains proper protein structure in acidic buffers required formaintaining lysosomal enzyme activities.

The collective data therefore show that chemical conjugation of IGF2peptide onto lysosomal enzymes (e.g., rhGAA and I2S) can indeedsignificantly increase their binding affinities for the IGF2/CI-MPRreceptor. This approach should theoretically be broadly applicable forchemical conjugation of variant IGF2 peptides onto any lysosomal proteinand other non-lysosomal proteins for improving their binding affinityfor the IGF2/CI-MPR.

Example 7

To assess the functional effects of IGF2 peptide for the cellular uptakeof exogenous lysosomal enzymes, the internalization of IGF2-conjugatedrbGAA (vIGF2-rhGAA) was evaluated in L6 rat skeletal muscle myoblasts.Briefly, L6 myoblasts were expanded in T-75 flasks to confluence in DMEMmedium containing 10% fetal bovine serum (FBS) at 37 C and a 5% CO2environment. The cells were harvested via trypsin/EDTA and plated in6-well tissue culture plates at a cell density of 3×10⁵ cells per welland incubated in DMEM/10% PBS medium. Two hours prior to the addition oflysosomal enzymes, the spent DMEM 10% FBS medium was replaced with 2.5ml uptake medium (Ham's F-12/10% FBS/2 mM PIPES, pH 6.7). UnconjugatedrhGAA and vIGF2-rhGAA were diluted to 0.5 mg/ml with 50 mM sodiumphosphate, pH 6.5/100 mM NaCl/0.05%>Polysorbate-80 and sterilizedthrough a 0.2 μm filter spin device (Costar). Unconjugated rhGAA wasadded to individual wells at final protein concentrations of 10-200 nMwhile vIGF2-rhGAA was added at 2-25 nM. To ensure that all wells had thesame volume and correct protein concentration, 50 mM sodium phosphate,pH 6.5/100 mM NaCl/0.05% Polysorbate-80 buffer was added so that thetotal volume of enzyme and buffer was 0.2 ml for each sample. As shownin FIG. 6B, the internalization of vIGF2-rhGAA was significantly betterthan unconjugated rhGAA at all protein concentrations tested. Theseresults revealed several important aspects about vIGF2-rhGAA: (1) theprotein structure of variant IGF2 peptide is sufficient for highaffinity binding to cell surface IGF2/CI-MPR receptors; (2) vIGF2-rhGAAwas efficiently internalized in L6 myoblasts and delivered to lysosomessince intracellular organelles were isolated with this protocol; (3)variant IGF2 peptide has low binding affinity to serum IGF bindingproteins (IGFBPs) as predicted since vIGF2-rhGAA was internalized in L6myoblasts rather than being bound to IGBPs in medium; (4) chemicalcoupling of variant IGF2 peptides did not alter rhGAA enzyme activity.

These results clearly show that chemical coupling of variant IGF2peptides onto rhGAA can significantly improve its binding affinity forthe IGF2/CI-MPR which directly translates into substantially improvedcellular uptake of the lysosomal enzyme in target cells. These datasuggest that vIGF2-rhGAA would be a superior ERT for the treatment ofPompe disease.

Example 8

To determine whether multiple IGF2 peptides were chemically conjugatedto lysosomal enzymes, sodium dodecylsulfate polyacrylamide protein gelelectrophoresis (SDS-PAGE) was utilized to separate proteins based ontheir size and the gel was subsequently stained with a modifiedCoomassie blue stain for visualization of protein bands. As shown inFIG. 7B, the molecular weight of recombinant wildtype human iduronidate2-sulfatase (I2S) was significantly increased from ˜80 kDa toapproximately 120 kDa after chemical conjugation of variant IGF2peptide. These data clearly show that multiple variant IGF2 peptidesmust have been coupled to I2S since the molecular weight of variant IGF2peptide is approximately only 8 kDa. The stained SDS-PAGE gel shows thatthere is a distribution of vIGF2-I2S species with varying amounts ofattached variant IGF2 peptide with an average of 5 attached peptides permolecule of I2S (corresponding to an increase of ˜40 kDa to attain theapproximate 120 kDa molecular weight on gel). Importantly, these dataalso highlight the potential of this approach for coupling multiple,different peptides onto the same lysosomal enzyme. For example, variantIGF2 peptides and other targeting peptides (e.g., peptides that areknown to be transported across the blood brain barrier (BBB)) could bechemically coupled to the same lysosomal enzyme for targeting thelysosomal enzyme to visceral tissues (via IGF2 peptide) and to the brainand central nervous system (via BBB-penetrating peptides). This approachtherefore has the potential to overcome the major limitations of currentERTs.

Example 9

SEQ ID NO: 1 represents the cDNA sequence for 8×His-tagged [(del1-4)-Arg6-Leu27-Arg65] IGF-2 peptide with an N-terminal extension linkerregion and a TEV protease recognition site (optimized for expression inE. coli).

SEQ ID NO: 1 atgggcagccaccaccaccatcatcaccaccacactagtgccggcgagaatctgtactttcagggcggtggtggtagcggcggtggtggtagccgtaccctgtgtggtggcgaattggttgatacgctgcaattcgtctgtggtgaccgcggtttcctgttctctcgtccggcgtcccgcgtgagccgtcgcagccgtggtatcgttgaagagtgctgttttcgtagctgcgacctggctctgctggaaacctattgcgcgaccccggcacgtagcgagtga

SEQ ID NO: 2: represents the amino acid sequence for variant IGF2peptide with the extension sequence.

SEQ ID NO: 2 NH2-GGGGSGGGGSRTLCGGELVDTLQPVCGDRGFLFSRPASRVSRRSRGIVEECCFRSCDLA LLETYCATPARSE-COOH

SEQ ID NO: 2 corresponds to a variant IGF2 peptide after removal ofN-terminal 8× His tag via TEV protease. This variant IGF2 peptide lacksresidues 1-4 such that the N-terminal serine residue corresponds toresidue 5 of WT IGF2. Arginine substituted for glutamic acid at position6 is known to substantially lower binding affinity of IGF2 peptide forserum IGF binding proteins (IGFBPs). Substitution of leucine fortyrosine at position 27 is known to substantially lower binding affinityof IGF2 peptide for insulin and IGF1 receptors. A conservativesubstitution of arginine for lysine at position 65 was utilized toenable chemical modification of only the extension linker region at theN-terminus for conjugation to lysosomal enzymes. The N-terminalextension region is represented by SEQ ID NO: 3.

SEQ ID NO: 3 GGGGSGGGThe N-terminal glycine residue in SEQ ID NO:3 is used for chemicalmodification for coupling to lysosomal enzymes.

Example 10

SEQ ID No: 5 represents the cDNA sequence for 8×His-[(del1-4)-Arg6-Leu27-Arg65] IGF-2 peptide that was optimized for expressionin E. coli.

SEQ ID NO:5:

SEQ ID NO: 6:

CCATGGGCTCAAGCCACCACCACCACCACCACCACCACAGCGGCGAGAACCTGTACTTTCAGAGCCGTACCTTGTGCGGTGGTGAGCTGGTGGATACTCTGCAATTTGTTTGCGGCGACCGCGGCTTCCTGTTCAGCCGTCCGGCGAGCCGTGTTTCCCGTCGTAGCCGTGGTATCGTCGAAGAGTGTTGTTTCCGCTCTTGTGACCTGGCGCTGCTGGAAACGTATTGCGCTACCCCGGCACGCTCGGAAGGTGGTGGCGGCAGCGGTGGTGGTAGCAAAGGTTGAGCGGCCGCATAAGCGGCCGC A TAAT

ORF for Optimized 8XHis-Modified IGF-2 Peptide:   3 atgggctcaagccaccaccaccaccaccaccaccacagcggcgag    M  G  S  S  H  H  H  H  H  H  H  H  S  G   E  48 aacctgtactttcagagccgtaccttgtgcggtggtgagctggtg N  L  Y  F  Q  S  R  T  L  C  G  G  E  L  V  93 gatactctgcaatttgtttgcggcgaccgcggcttcctgttcagcD  T  L  Q  F  V  C  G  D  R  G  F  L  F  S 138 cgtccggcgagccgtgtttcccgtcgtagccgtggtatcgtcgaaR  P  A  S  R  V  S  R  R  S  R  G  I  V  E 183 gagtgttgtttccgctcttgtgacctggcgctgctggaaacgtatE  C  C  F  R  S  C  D  L  A  L  L  E  T  Y 228tgcgctaccccggcacgctcggaaggtggtggcggcagcggtggtC  A  T  P  A  R  S  E  G  G  G  G  S  G  G 273  ggtagcaaaggttga 287

  *

SEQ ID NO: 6 represents the amino acid sequence for a variant IGF2peptide with the extension sequence.

SEQ ID NO: 6: NH2- SRTLCGGELVDTLQFVCGDRGFLFSRPASRVSRRSRGIVEECCFRSCDLALLETYCATPARSEGGGGSGGGSKG-COOH

SEQ ID NO: 6 corresponds to a variant IGF2 peptide after removal ofN-terminal 8×His tag via TEV protease. The TEV protease recognition siteis underlined above. This variant JGF2 peptide lacks residues 1-4 suchthat the N-terminal serine residue corresponds to residue 5 of WT IGF2.Arginine substituted for glutamic acid at position 6 is known tosubstantially lower binding affinity of IGF2 peptide for serum IGFbinding proteins (IGFBPs). Substitution of leucine for tyrosine atposition 27 is known to substantially lower binding affinity of JGF2peptide for insulin and JGF1 receptors. A conservative substitution ofarginine for lysine at position 65 was utilized to incorporate a lysineresidue within extension region to enable chemical modification andconjugation to lysosomal enzymes. The extension region is represented bySEQ ID NO: 7.

SEQ ID NO: 7: GGGGSGGGSKG

The lysine residue in SEQ ID NO:7 is used for chemical modification forcoupling to lysosomal enzymes.

What is claimed:
 1. A conjugate, comprising: a variant IGF-2 peptidechemically conjugated to a recombinant human lysosomal the variant IGF-2peptide comprises SEQ ID NO:2 or SEQ ID NO:6 or, comprises one or moreof the following modifications with respect to SEQ ID NO: 8:substitution of arginine for glutamic acid at position 6; deletion ofamino acids 1-4 and 6; deletion of amino acids 1-4, 6 and 7; deletion ofamino acids 1-4 and 6 and substitution of lysine for threonine atposition 7; deletion of amino acids 1-4 and substitution of glycine forglutamic acid at position 6 and substitution of lysine for threonine atposition 7; substitution of leucine for tyrosine at position 27;substitution of leucine for valine at position 43; substitution ofarginine for lysine at position 65; and the IGF-2 peptide furthercomprises an affinity tag and/or a linker extension region.
 2. Theconjugate of claim 1, wherein the recombinant human lysosomal enzymecomprises one or more modified lysine residues, a chemically modifiedN-terminus, or a combination thereof.
 3. The conjugate of claim 1,wherein the recombinant human lysosomal enzyme is human acida-glucosidase (rhGAA).
 4. The conjugate of claim 1, wherein the chemicalconjugation is via a cross linking agent comprises an aminoreactivebifunctional cross linker.
 5. The conjugate of claim 1, wherein thechemical conjugation is via a cross linking agent comprisesN-succinimidyl 6-hydrazinonicotinate acetone (S-Hynic).
 6. The conjugateof claim 1, wherein the chemical conjugation is via a cross linkingagent comprises sulfo-Nhydroxysuccinimide ester-phosphine(sulfo-NHS-phosphine).
 7. The conjugate of claim 1, wherein the-chemicalconjugation is via a cross linking agent comprises N-hydroxysuccinimideester-tetraoxapentadecane acetylene (NHS-PEG4-acetylene).
 8. Theconjugate of claim 1, wherein the chemical conjugation is via acrosslinking agent comprises heterobifunctional cross linkers selectedfrom difluorocyclooctyne (DIFO) and dibenzocyclooctyne (DIBO).
 9. Amethod for treating a subject suffering from a lysosomal storagedisease, the method comprising administering to the subject theconjugate of claim 1 in an amount sufficient to treat the lysosomalstorage disease.
 10. The method of claim 9, wherein the lysosomalstorage disease is at least one of the following: Pompe Disease, FabryDisease, and Gaucher Disease, MPS I, MPS II, MPS VII, Tay Sachs,Sandhoff, a-mannosidosis, and Wohlman disease.
 11. The method of claim10, wherein the lysosomal storage disease is Pompe Disease.
 12. Themethod of claim 10, wherein the lysosomal storage disease is FabryDisease.
 13. The method of claim 10, wherein the lysosomal storagedisease is Gaucher Disease.
 14. The conjugate of claim 1, wherein theIGF-2 peptide comprises SEQ ID NO:2.
 15. The conjugate of claim 1,wherein the IGF-2 peptide comprises SEQ ID NO:6.
 16. The conjugate ofclaim 1, wherein the IGF-2 peptide further comprises a linker, whereinsaid linker is 5 to 20 amino acid residues in length.
 17. The conjugateof claim 16, wherein said linker is about 10 amino acid residues inlength.
 18. The conjugate of claim 16, wherein said linker comprises SEQID NO:3.
 19. The conjugate of claim 16, wherein said linker comprisesSEQ ID NO:7.